Long Noncoding RNA MALAT1 Regulates Cancer Glucose

Published OnlineFirst March 26, 2019; DOI: 10.1158/0008-5472.CAN-18-1432

Cancer Metabolism and Chemical Biology Research

Long Noncoding RNA MALAT1 Regulates Cancer Glucose Metabolism by Enhancing mTOR-Mediated Translation of TCF7L2 Pushkar Malakar1, Ilan Stein2,3, Amijai Saragovi2,3, Roni Winkler4, Noam Stern-Ginossar4, Michael Berger2,3, Eli Pikarsky2,3, and Rotem Karni1

Abstract

Reprogrammed glucose metabolism of enhanced aerobic expression regulated TCF7L2 mRNA association with heavy glycolysis (or the Warburg effect) is known as a hallmark of polysomes, probably through the TCF7L2 50-untranslated cancer. The roles of long noncoding RNAs (lncRNA) in regu- region (UTR), as determined by polysome fractionation lating cancer metabolism at the level of both glycolysis and and 50UTR-reporter assays. Knockdown of TCF7L2 in gluconeogenesis are mostly unknown. We previously showed MALAT1-overexpressing cells and HCC cell lines affected that lncRNA metastasis-associated lung adenocarcinoma tran- their metabolism and abolished their tumorigenic poten- script 1 (MALAT1) acts as a proto-oncogene in hepatocellular tial, suggesting that the effects of MALAT1 on glucose carcinoma (HCC). Here, we investigated the role of MALAT1 metabolism are essential for its oncogenic activity. Taken in regulating cancer glucose metabolism. MALAT1 upregu- together, our findings suggest that MALAT1 contributes to lated the expression of glycolytic genes and downregulated HCC development and tumor progression by reprogram- gluconeogenic enzymes by enhancing the translation of the ming tumor glucose metabolism. metabolic transcription factor TCF7L2. MALAT1-enhanced TCF7L2 translation was mediated by upregulation of SRSF1 Significance: These findings show that lncRNA MALAT1 and activation of the mTORC1–4EBP1 axis. Pharmacological contributes to HCC development by regulating cancer glucose or genetic inhibition of mTOR and Raptor or expression of a metabolism, enhancing glycolysis, and inhibiting gluconeo- hypophosphorylated mutant version of eIF4E-binding protein genesis via elevated translation of the transcription factor (4EBP1) resulted in decreased expression of TCF7L2. MALAT1 TCF7L2.

Introduction the splicing factor SRSF1 and mTORC1 activation (8). Further- more, we showed that mTORC1 activation is required for Long noncoding RNAs (lncRNA) constitute a large class of MALAT1-mediated tumorigenesis (8). mRNA-like transcripts, greater than 200 nucleotides with no Both the Wnt and mTOR signaling pathways have been shown protein coding capability (1). In the past few years, several to play an important role in altering the glucose metabolic lncRNAs have been shown to play a role in cancer by promoting program in cancers (9, 10). Altered glucose metabolism is one proliferation, invasion and metastasis (2–4). LncRNAs have been of the first identified hallmarks of cancer (11), discovered by Otto shown to regulate almost every step of gene expression (5). Warburg in the late 1920s (12). Cancer cells predominantly carry MALAT1 was one of the first lncRNAs to have a designated role out glycolysis in the cytosol rather than oxidative phosphorylation in cancer (4, 6). MALAT1 is highly conserved among mammals, through the TCA cycle in the mitochondria (13). It is generally approximately 7Kb in length and highly abundant (7). Previous- believed that in most cancers, oncogenic lesions are largely the ly, we showed that MALAT1 acts as a proto-oncogene in hepato- cause of enhanced glycolysis and the "Warburg effect" (14). cellular carcinoma through Wnt pathway activation, induction of c-MYC, a downstream target of Wnt signaling, was shown to play an important role in the regulation of glycolysis in cancer 1Department of Biochemistry and Molecular Biology, Institute for Medical cells (15). Glucose metabolism genes were shown to be directly Research Israel Canada (IMRIC), Hebrew University-Hadassah Medical School, regulated by c-MYC. The key modulator of the canonical Wnt Jerusalem, Israel. 2The Lautenberg Center for Immunology and Cancer Research, signaling pathway is the bipartite transcription factor b-Cat Institute for Medical Research Israel Canada (IMRIC), Jerusalem, Israel. 3Depart- (b-catenin)/TCF, formed by b-catenin and a member of the TCF ment of Pathology, Hebrew University—Hadassah Medical School, Jerusalem, family (TCF-1, LEF-1, TCF-3 and TCF-4/TCF7L2; ref. 16). TCF7L2 Israel. 4Department of Molecular Genetics, Weizmann Institute of Science, was shown to be an effector of the Wnt signaling pathway and Rehovot, Israel. binds directly to multiple genes that are important in regulating Note: Supplementary data for this article are available at Cancer Research glucose metabolism. Moreover, genome-wide association studies Online (http://cancerres.aacrjournals.org/). (GWAS) have identified SNPs in the TCF7L2 gene associated with Corresponding Author: Rotem Karni, Hebrew University—Hadassah Medical obesity and diabetes (17). mTOR activation regulates glucose School, Ein Karem, 91120, Jerusalem, Israel. Phone: 972-2-675-8289; Fax: 972-2- metabolism through activation of HIF1a. HIF1a is a transcription 675-7379; E-mail: [email protected] factor that is known to induce the expression of at least 9 glycolytic doi: 10.1158/0008-5472.CAN-18-1432 enzymes, thereby regulating glucose metabolism in many can- 2019 American Association for Cancer Research. cers (18). Several lncRNAs have been shown to regulate, or to be

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LncRNA RNA MALAT1 Regulates Cancer Glucose Metabolism

regulated by, the Wnt and mTOR signaling pathways (19, 20). against Luciferase (Dharmacon Thermo Scientific) or siRNA Uni- Even though lncRNAs have been shown to affect cancer initiation versal Negative Control (Sigma) was used as a control at specified and progression, only a handful of studies have focused on the concentrations. Lipofectamine 2000 reagent (Invitrogen) was involvement of lncRNAs in cancer glucose metabolism at the level used for transfection as per the manufacturer's instructions. of glycolysis (21, 22). Gluconeogenesis is essentially the reverse of the glycolysis pathway and usually occurs in the liver when blood qRT-PCR glucose levels drop and the liver regenerates glucose, sending it to Total RNA was extracted with TRI Reagent (Sigma), and 1 mgof other tissues (23). There is a single report describing the role of total RNA was reverse transcribed using M-MLV reverse transcrip- lncRNAs in cancer development through regulating gluconeogen- tase (Promega) after DNase treatment (Promega). qPCR was esis. lncRNA Nur77 was shown to suppress HCC through upre- performed on the cDNA using SYBR Green Mix (Roche) and gulating gluconeogenesis (24). There are no reports describing the CFX96 (Bio-Rad) real-time PCR machine. Primer list is supplied role of MALAT1 in cancer glucose metabolism at the level of in Supplementary Table S1. glycolysis or gluconeogenesis. Several studies have shown gluconeogenesis to be downregulated in HCC (25, 26). However, the Immunoblotting regulation of both glycolysis and gluconeogenesis by lncRNAs in Cells were lysed in Laemmli buffer and analyzed for total HCC development or progression has not been reported. protein concentration. Twenty micrograms of total protein In this study, we investigated the roles of MALAT1 in regulating from each cell lysate were separated by SDS-PAGE and transferred glucose metabolism of HCC cancer cells and found that MALAT1 to a polyvinylidene difluoride (PVDF) membrane. Primary enhanced aerobic glycolysis and repressed gluconeogenesis. We antibodies used were TCF7L2 EP20334 (1:10,000; Abcam), further discovered that MALAT1 regulates glycolytic gene expres- GAPDH (1:5,000; Sigma), b-catenin (1:2,000; Sigma), b-actin sion through increased translation of transcription factor TCF7L2. (1:2,000; Sigma). a-Tubulin (1:1,000; Santa Cruz Biotechnol- We demonstrate, both pharmacologically and genetically, that ogy), b-Tubulin (1:2000;Sigma). SRSF1 (AK96 culture super- TCF7L2 upregulation is mediated by mTORC1 activation of cap- natant 1:300), T7 Tag (1:5,000; BD Transduction laboratories), dependent translation. MALAT1-mediated tumorigenesis is mTOR (1:1,000; Cell Signaling Technology), Raptor (1:1,000; dependent on TCF7L2. In addition, using Mdr2 / mice liver Cell Signaling Technology), p4EBP1 (1:1,000; Cell Signaling tumor samples, we show elevated levels of MALAT1, nuclear Technology), Total 4EBP1 (1:1,000; Cell Signaling Technolo- TCF7L2 and glycolytic gene expression, and decreased expression gy). Secondary antibodies used were HRP-conjugated goat anti- of gluconeogenic gene expression, suggesting a positive correla- mouse, goat anti-rabbit, donkey anti-goat IgG (HþL; 1:10,000; tion with glycolysis and a negative correlation with gluconeogen- The Jackson Laboratory). esis. Thus, we present here a novel function for MALAT1 in tumorigenesis and provide a previously unappreciated mecha- Colony formation assay nism by which cancer cells switch to aerobic glycolysis, repressing Cells were seeded in 6-well plates (1,000 cells/well) and grown gluconeogenesis, during cancer progression. This is the first report for 10 days. After fixation with 2.5% glutaraldehyde, the plates showing the regulation of TCF7L2 by mTORC1-mediated cap- were washed three times. Fixed cells were then stained with dependent translation and suggests that the mTORC1 pathway methylene blue solution (1% methylene blue in 0.1 mol/L borate can regulate Wnt signaling through TCF7L2 translation. buffer, pH 8.5) for 60 minutes at room temperature. Plates were photographed after extensive washing and air drying (28).

Materials and Methods Anchorage-independent growth Cell culture Colony formation in soft agar was assayed as described previ- PHM-1 cells are mouse liver progenitor cells derived from ously [41]. After 14 to 21 days, colonies from 10 different fields in embryonic day 18 fetal livers from TP53 / mice and immor- each of two wells were counted for each treatment and the average talized with MSCV-based retroviruses expressing MYC-IRES- number of colonies per well was calculated. The colonies were GFP (27). PHM-1, FLC4, and HepG2 cells were grown in stained and photographed under a light microscope at 10 DMEM supplemented with 10% FCS, 0.1 mg/mL penicillin, magnification (28). and 0.1 mg/mL streptomycin. All cell lines have been tested and authenticated using STR loci plus Amelogenin for gender iden- Lactate assay tification for human cell line authentication by the Biosynthesis Cells (2 105) were seeded in 6-well culture plates. The cells DNA Identity Testing Centre. were trypsinized 48 hours after culture or siRNA treatment. Cells were homogenized in the presence of lactate assay buffer and Stable cell lines centrifuged at 13,000 g for 10 minutes. Lactate quantification pCD513B1 empty (System Biosciences) and pCD513B1- was performed using commercially available lactate assay kit hMALAT1 lentiviruses were prepared using the manufacturer's (Abcam, ab65330) in a 96-well plate as per the manufacturer's instructions. These viruses were used to infect PHM-1 cells. Cells instructions. Lactate levels were measured using a plate reader at were selected by the addition of puromycin (2 mg/mL) for 72 to an optical density of 570 nm (29). Lactate levels were normalized 96 hours. In the case of infection with MLP-puro-shRNA viruses, to total cellular protein concentration. cells were selected with puromycin (2 mg/mL) for 96 hours. Glucose secretion assay siRNA treatment HepG2 and FLC4 cells were cultured and treated with siRNAs. Double-stranded siRNAs (Sigma) were used at specified con- After 48 hours of siRNA treatment, the medium was replaced with centrations to deplete MALAT1 or TCF7L2 from cells. siRNAs DMEM containing 0.1% serum for 16 hours. Cells were washed

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twice with PBS to remove glucose and then incubated for 6 hours System according to the protocol provided by Promega and in glucose production assay medium (glucose and phenol red-free Infinite M200 PRO. Renilla activity was used to normalize for DMEM containing 2 mmol/L sodium pyruvate, 20 mmol/L transfection efficiency. Firefly-luciferase mRNA expression was sodium lactate, 2 mmol/L L-glutamine and 15 mmol/L HEPES). also measured as an additional control for luciferase activity. Medium (200 mL) was sampled for measurement of glucose concentration. Glucose level quantification was performed using Glucose uptake in FLC4 cells a commercially available glucose assay kit (Amplex Red Glucose FLC4 cells were treated with siRNAs targeted against luciferase Assay Kit, ThermoFisher Scientific) in a 96-well plate. Glucose or MALAT1 for 48 hours. Subsequently, they were treated with levels were normalized to total cellular protein concentration. medium without glucose for 16 hours and then exposed to 2NBDG (a fluorescent derivative of glucose) for 30 minutes. Mice 2NBDG fluorescence was recorded using flow cytometry. All animal experiments were performed in accordance with the / institutional animal care and use committee. Mdr2 mice (30) Statistical analysis fi were bred and maintained in speci c pathogen-free conditions. Error bars for all data represent SDs from the mean. P values were calculated using two-tailed type 2 Student t tests except for Immunohistochemistry a few cases where tail one type 2 Student t test was used. m Immunohistochemistry for TCF7L2 was performed on 5- m Statistical significance is displayed as , P < 0.05; , P < 0.01; fi fi formalin- xed paraf n-embedded sections. After citrate-based and , P <0.001. antigen retrieval (Vector Labs # H-3300), endogenous perox- idase was blocked by 3% H2O2. Slides were incubated with primary antibody (a-TCF7L2, Abcam # ab76151), washed, and incubated with anti-Rabbit-HRP ImmPRESS Reagent (Vector Results Labs # MP-7401). Slides were developed with the HRP substrate MALAT1 affects glucose metabolism in immortalized and diaminobenzidine (Thermo Scientific) and counterstained with cancerous liver cells, promoting aerobic glycolysis hematoxylin. One of the first identified hallmarks of cancer is altered glucose metabolism (32). Tumor cells enhance glycolysis even in the Polysome profiling presence of oxygen and in many cases reduce oxidative phos- Polysome profile analysis was carried out as described previ- phorylation (13). In addition, many oncogenes enhance glycol- ously (31). Briefly, cells were cultured in 10cm dishes. Before ysis by alternative mechanisms (18). We previously found that the harvesting cells were treated with cyclohexamide (20 mL CHX lncRNA MALAT1 acts as a proto-oncogene in HCC development from 50 mg/mL stock) for 3 minutes. Then cells were washed and activates the mTORC1 pathway (8). The mTORC1 pathway twice with cold PBS containing 50 mg/mL cycloheximide, col- affects tumor metabolism by several mechanisms and specifically lected, and lysed in a 250 mL of lysis buffer [Lysis Buffer (5 mL): glucose metabolism (33). Thus, we sought to examine the effect of 250 mL 20% Triton (RNAse free) þ 4.75 mL Polysome buffer þ MALAT1 on glucose metabolism in HCC cancer cells. One of the 60 mL DNAse (120 U)]. Lysates were loaded onto 10% to 50% characteristics of hepatocytes is their ability to produce glucose by sucrose density gradients prepared in polysome buffer. [Polysome gluconeogenesis, to supply glucose to the body when blood Buffer (20 mL): 250 mL of 1 mol/L Tris pH 7, 150 mL of 1 mol/L Tris glucose levels drop (23). This process acts in a reverse pathway pH 8, 600 mL of 5 mol/L NaCl, 100 mL of 1 mol/L MgCl2, 40 mLof to glycolysis. We measured lactate production as a measure for CHX (50 mg/mL in EtOH), 20 mL of DTT (1 mol/L) and 18.84 mL glycolysis in PHM-1 cells either overexpressing or knocked-down of DEPC Water]. Extracts were fractionated for 3 hours at for MALAT1. Lactate production was measured by the intracellular 35,000 rpm at 4C in a Beckman rotor, and the gradients were lactate content. Overexpression of MALAT1 led to enhanced recovered in 12 fractions using gradient fractionators. RNA was lactate production (Fig. 1A and B). Conversely, transient knock- extracted from each fraction. Translational status of TCF7L2 down of MALAT1 by siRNAs resulted in reduced lactate produc- mRNA on polysome fractions was determined by qRT-PCR. tion (Fig. 1C and D). These data suggest that MALAT1 expression regulates glucose metabolism in PHM-1 cells by enhancing gly- Luciferase reporter assay colysis. To eliminate possible effects of cell proliferation or 507 bp of human TCF7L2, 50-untranslated region (UTR) cellular density on glucose metabolism, we examined glucose upstream of the start codon, were amplified from FLC4 cells uptake at a single cell level. Cells were labeled with fluorescent cDNA by RT-PCR using a forward primer with a KpnI restriction glucose (2NBDG) and glucose uptake was measured by flow site and a reverse primer with a XhoI restriction site and subcloned cytometry. We found that glucose uptake was lower following into the KpnI and XhoI restriction sites of the pSG5 Luc plasmid. MALAT1 knockdown in HCC FLC4 cells (Supplementary The insert was verified by sequencing. pSG5 Luc Plasmid was a Fig. S1A–S1B). In previously performed RNA-seq analysis on kind gift from Prof. Fatima Gebauer, Center for Genomic Regu- PHM-1 cells overexpressing MALAT1, we detected increased lation (CRG), Barcelona. PHM-1, HepG2 and FLC4 cells were expression of several glycolytic genes (8). To confirm the tran- seeded in 6-well plates (2 105 cells/well) under standard scriptional regulation of the glucose metabolism program in conditions. After 24 hours, cells were transfected with MALAT1 hepatocytes by MALAT1 we validated several of the upregulated siRNAs using Lipofectamine 2000. After another 48 hours these genes. In agreement with enhanced glycolysis, the expression of cells were further transfected, using polyethylenimine, with 2 mg several glycolytic enzymes was upregulated in cells overexpressing of TCF7L2-50UTR-Firefly construct and 0.5mg pRenilla construct MALAT1 (Fig. 1E–G) and reduced by knockdown of MALAT1 per well. Forty-eight hours later, the cells were harvested and (Supplementary Fig. S1C–S1E). These data suggest that MALAT1 luciferase activity was analyzed using Dual-Glo Luciferase Assay expression promotes glycolytic metabolism in cancer cells.

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LncRNA RNA MALAT1 Regulates Cancer Glucose Metabolism

A B 3 Lactate 1.5 MALAT1 * 2.5 1.2 2 0.9 1.5 0.6 1

0.3 Relative output 0.5 0 Relative expression 0 Empty MALAT1 Empty MALAT1 PHM-1 PHM-1 D C 1.2 1.2 Lactate MALAT1 1 1 *** 0.8 0.8 *** 0.6 * 0.6 * 0.4 0.4 Relative output

Relative expression 0.2 0.2

0 0 siLuciferase siMALAT1#1 siMALAT1#2 siLuciferase siMALAT1#1 siMALAT1#2 PHM-1 MALAT1 PHM-1 MALAT1 EF

GLUT1

HK2

ENO1

PKM2

GAPDH G PHM-1 4 GLUT1 5 HK2 *** 2.5 ENO1 ** 3 PKM2 3.5 * 4 2 2.5 *** 3 2 2.5 3 1.5 2 1.5 2 1 1.5 1 1 1 0.5 0.5 0.5 Relative expression 0 0 0 0 Empty MALAT1 Empty MALAT1 Empty MALAT1 Empty MALAT1 PHM-1 PHM-1 PHM-1 PHM-1

Figure 1. MALAT1 affects cancer glucose metabolism. A, qRT-PCR of PHM-1 cells stably expressing hMALAT1 or an empty vector. B, Extracellular lactate production was measured in cells described in A using a lactate assay kit (n ¼ 3). C, PHM-1 cells overexpressing MALAT1 knocked down for MALAT1 by siRNAs (siMALAT#1, #2) were analyzed by qRT-PCR. D, Extracellular lactate production was measured in cells described in D using a lactate assay kit (n ¼ 3). E, Schematic representation of the glycolytic and gluconeogenetic pathways of glucose metabolism. The enzymes marked in red were selected for gene expression analysis. F, A gel image of semiquantitative RT-PCR of glycolytic gene expression in PHM-1 cells described in A. G, Expression of genes in the glucose metabolic pathway in cells described in A measured by qRT-PCR. All samples were normalized to GAPDH mRNA levels. Error bars, SD (n ¼ 3). Student t test was used. , P < 0.05; , P < 0.01; , P < 0.001.

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ACB 1.2 MALAT1 Glucose secretion 1.2 Lactate 4 ** 1 3.5 0.8 3 0.8 *** 2.5 * 0.6 *** ** *** 2 0.4 1.5 0.4

1 Relative output Relative expression 0.2 Relative abundance 0.5 0 0 0

HepG2 HepG2 HepG2

DEF G6PC PCK1 7 3.5 ** * *** 6 3 5 ** 2.5 4 2 3 1.5 2 1 1 Relative expression 0.5 Relative expression 0 0

HepG2 HepG2

Figure 2. MALAT1 negatively regulates gluconeogenesis in HCC cells. A, HepG2 cells were transfected with either MALAT1 siRNA (siMALAT#1, #2) or control siRNA (siLuciferase). MALAT1 RNA levels were analyzed by qRT-PCR. B. Cellular glucose secretion was measured in cells described in A using a glucose assay kit (n ¼ 3). C, Extracellular lactate production was measured in cells described in A using a lactate assay kit (n ¼ 2). D, Schematic representation of the glycolytic and gluconeogenetic pathways. The enzymes marked in blue are involved in gluconeogenesis. E and F, mRNA expression of the G6PC (E) and PCK1 (F) genes in the gluconeogenesis pathway in cells described in A. All samples were normalized to actin or GAPDH mRNA levels. Error bars, SD (n ¼ 3). Student t test was used. , P < 0.05; , P < 0.01; , P < 0.001.

MALAT1 negatively affects gluconeogenesis MALAT1 controls TCF7L2 expression at the protein level Gluconeogenesis is a major component of glucose metabolism The transcription factor TCF7L2 was shown to modulate glu- in normal liver cells, regulating whole-body glucose homeosta- cose homeostasis in the liver (36, 37). Moreover, it has been sis (34). In HCC, gluconeogenesis plays a tumor-suppressive role shown that TCF7L2 negatively regulates gluconeogenesis (38). opposing aerobic glycolysis and preventing the "Warburg Although the role of TCF7L2 in the Wnt signaling pathway is well effect" (35). To examine the effect of MALAT1 on gluconeogenesis studied, its role in modulating glucose metabolism is less well in HCC, we knocked-down MALAT1 in HCC cell lines and characterized, and in some cases conﬂicting (39). Thus, we exam- examined gluconeogenic gene expression. Expression of gluco- ined the regulation of TCF7L2 by MALAT1. To probe the potential neogenic genes is downregulated in HCC compared with normal mechanism by which MALAT1 regulates TCF7L2, we examined hepatocytes (25, 26). We found that transient knockdown of the effects of MALAT1 manipulation on the expression of TCF7L2. MALAT1 by siRNAs in HepG2 cells (Fig. 2A) and FLC4 cells We detected no signiﬁcant change in the mRNA levels of TCF7L2 (Supplementary Fig. S2A) resulted in increased glucose secretion in response to MALAT1 overexpression or knockdown (Fig. 3A). (Fig. 2B; Supplementary Fig. S2B) and reduced lactate production In contrast with these results, western blot analysis showed (Fig. 2C; Supplementary Fig. S2C). Transient knockdown of that MALAT1 overexpression resulted in enhanced TCF7L2 pro- MALAT1 in these cells also resulted in increased expression of tein expression (Fig. 3B). Transient knockdown of MALAT1 did gluconeogenic genes, G6PC and PCK1 (Fig. 2D–F; Supplementary not change TCF7L2 mRNA level (Fig. 3C), but resulted in Fig. S2D and S2E). decreased protein expression of TCF7L2 (Fig. 3D; Supplementary

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LncRNA RNA MALAT1 Regulates Cancer Glucose Metabolism

2 3.5 A TCF7L2 B 3 *** 1.6 2.5 1.2 TCF7L2 2 1.5 0.8 β-Catenin 1 0.4

Relative intensity 0.5 Relative expression Tubulin 0 0 Empty MALAT1 PHM-1 Empty MALAT1 PHM-1 PHM-1 C D 1.2 2 TCF7L2 1 1.6 0.8 * 1.2 TCF7L2 0.6 *** 0.8 0.4 b-Actin 0.4 Relative intensity 0.2 Relative expression GAPDH 0 0 PHM-1 MALAT1

PHM-1 MALAT1 PHM-1 MALAT1

E 0.7 * F 0.7 ** ** Empty siLuciferase 0.6 0.6 MALAT1 siMALAT1 0.5 0.5

0.4 0.4 * 0.3 0.3 * 2 mRNA 2 mRNA Fraction L2 mRNA L2 mRNA Fraction 0.2 **

0.2 7 0.1

0.1 TCF TCF7L 0 0 Free Light Heavy Free Light Heavy PHM-1 PHM-1 G 1.2 H 2.5 1 Luciferase mRNA 2 0.8 *** 1.5 0.6 ***

activity 1 0.4

0.5 0.2 Normalized luciferase Relative expression Relative 0 0 siControl siMALAT1#1 siMALAT1#2 siControl siMALAT1#1 siMALAT1#2 PHM-1 PHM-1

Figure 3. MALAT1 upregulates TCF7L2 translation. A, TCF7L2 mRNA expression in PHM-1 cells stably expressing hMALAT1 or an empty vector analyzed by qRT-PCR. B, Left, TCF7L2 protein levels in cells described in A analyzed by Western blot. Right, quantiﬁcation of TCF7L2 protein levels (n ¼ 4). C, TCF7L2 mRNA levels in PHM-1 cells overexpressing MALAT1 treated with the indicated siRNAs, as determined by qRT-PCR. D, Left, TCF7L2 protein levels in cells described in C were determined by Western blot. Right, quantiﬁcation of TCF7L2 protein levels (n ¼ 3). E, Relative distribution of TCF7L2 mRNA across the polysome fractions in PHM-1 cells stably expressing hMALAT1 or an empty vector. F, Relative distribution of TCF7L2 mRNA across the polysome fractions in cells transfected with either MALAT1 siRNA or control siRNA (siLuciferase). G, PHM-1 cells were transfected with MALAT1 siRNAs (siMALAT1#1, #2) or siControl. Luciferase activity (fold change compared with siControl) produced after transfection with luciferase construct containing the WT TCF7L2 50UTR was measured. Luciferase activity was normalized to Renilla expression. H, The graph shows the luciferase transcript expression measured by qRT-PCR. All bars show the average of two to three experiments. Error bars, SD. Student t test was used. , P < 0.05; , P < 0.01; , P < 0.001.

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Fig. S3A–S3B). These results suggest that the regulation of TCF7L2 glycolytic genes (Supplementary Fig. S4B–S4E). These data sug- by MALAT1 is post-transcriptional. To investigate the mechanism gest that the phosphorylation status of 4EBP1 is important for of TCF7L2 translational regulation by MALAT1, we characterized regulation of TCF7L2 protein expression and consequently its polysome-associated TCF7L2 mRNA in MALAT1-manipulated downstream targets. cells. Overexpression of MALAT1 resulted in enhanced association of TCF7L2 mRNA with heavy polysomal fractions and MALAT1 regulates TCF7L2 translation through SRSF1 reduced association with light and free polysomal fractions MALAT1 was shown to activate the mTOR pathway by enhanc- (Fig. 3E). Furthermore, transient knockdown of MALAT1 resulted ing the expression and function of the splicing oncoprotein in increased association of TCF7L2 to free and light ribosome SRSF1 (8). It was shown previously that SRSF1 can activate mTOR fractions and decreased association of TCF7L2 mRNA to heavy and protein translation. To investigate the molecular mechanism ribosome fractions (Fig. 3F). This result suggests that MALAT1 by which MALAT1 (which is a nuclear lncRNA), regulates the regulates TCF7L2 translation. Translation initiation is mediated in translation of TCF7L2 in the cytoplasm, we examined the regu- many cases through the 50UTR, which can contain secondary RNA lation of TCF7L2 by SRSF1. Stable knockdown of SRSF1 in HepG2 structures or upstream open reading frames (ORF) that inhibit cells resulted in reduced protein expression of TCF7L2 (Fig. 5A). translation (40). To examine whether MALAT1 regulates TCF7L2 Furthermore, knockdown of SRSF1 in PHM-1 cells resulted in translation through the TCF7L2 50UTR, we subcloned TCF7L2 reduced protein expression of TCF7L2 (Supplementary Fig. S5A). 50UTR upstream of a luciferase reporter construct and measured We detected no significant change in the mRNA levels of TCF7L2 luciferase protein and mRNA levels following transient MALAT1 in response to SRSF1 knockdown in HepG2 and PHM-1 cells knockdown. Knockdown of MALAT1 lead to a significant reduc- (Fig. 5B; Supplementary Fig. S5B). Ribosome fractionation tion in luciferase activity from the TCF7L2 50UTR luciferase showed reduced binding of TCF7L2 mRNA to the heavy polysome reporter whereas the mRNA levels of luciferase were not affected fraction and elevated binding to the light polysome fraction upon (Fig. 3G and H; Supplementary Fig. S3C and S3D). This result SRSF1 knockdown (Fig. 5C). These results suggest that SRSF1 suggests that MALAT1 regulates TCF7L2 translation, at least partly, regulates the expression of TCF7L2 post-transcriptionally by through TCF7L2 50UTR. regulating its translation in hepatocellular carcinoma cells, a phenomenon seen for other proteins (45, 46). TCF7L2 translation is regulated by mTORC1 We have previously shown that MALAT1 upregulation activates TCF7L2 mediates the effects of MALAT1 on glucose metabolism the mTORC1 pathway. This was evident from the increased Silencing of TCF7L2 protein levels in hepatocytes leads to an phosphorylation of eIF4E binding protein (4EBP1) in PHM-1 increase in glucose output associated with elevated expression of cells overexpressing MALAT1, whereas knockdown of MALAT1 in multiple gluconeogenic genes (36, 47). TCF7L2 was shown to be these cells resulted in decreased phosphorylation of 4EBP1 (8). overexpressed and contribute to the malignant phenotype in mTORC1 regulates numerous components involved in protein HCC (48). TCF7L2 is an important mediator of the Wnt signaling synthesis, ranging from initiation and elongation factors to the pathway, a signal transduction pathway that directly contributes biogenesis of ribosomes themselves (41). mTORC1 promotes to the regulation of cellular metabolism. Because we observed that protein synthesis largely through the phosphorylation of two key knockdown of MALAT1 resulted in increased expression of glu- effectors, p70 S6 Kinase 1(S6K1) and 4EBP1 (41). We took three coneogenic genes (Fig. 2E and F), we decided to examine whether different approaches to assess the importance of mTORC1 acti- the effect of MALAT1 on glucose metabolism is mediated via vation in MALAT1-mediated regulation of TCF7L2 protein expres- TCF7L2. We introduced siRNAs targeting TCF7L2 into PHM-1 sion. First, we used the mTOR inhibitor rapamycin to block mTOR MALAT1 cells (Fig. 6A and B). Knockdown of TCF7L2 reduced catalytic activity as part of mTORC1. Increased expression of lactate production by MALAT1-overexpressing cells (Fig. 6C). TCF7L2 in MALAT1-overexpressing PHM-1 cells was reduced by Similarly, transient knockdown of TCF7L2 in HepG2 cells treatment of cells with rapamycin (Fig. 4A). Second, we used increased glucose secretion and reduced lactate production (Sup- shRNAs to knockdown either mTOR itself or Raptor, distinctive plementary Fig. S6A–S6C). This suggests that TCF7L2 is involved components of mTORC1. Knockdown of either of these factors in MALAT1-mediated glucose metabolism. Next, we examined reduced protein expression of TCF7L2 in PHM-1 cells (Fig. 4B and glycolytic gene expression following transduction of PHM-1 cells C). Thirdly, we overexpressed a mutant 4EBP1, 4EBP1-5A, in overexpressing MALAT1 with lentiviruses encoding shRNAs which the five known phosphorylation sites were replaced with against TCF7L2 (Fig. 6D). TCF7L2 knockdown reduced glycolytic alanine (42). Hyperphosphorylation of 4EBP1 is known to lead to gene expression in these cells (Fig. 6E–H). In HepG2 cells, TCF7L2 activation of cap-dependent translation. This mutant cannot be knockdown increased the expression of gluconeogenic genes phosphorylated and binds constitutively to eIF4E, thus inhibiting G6PC and PCK1 (Supplementary Fig. S6D and S6E). Collectively, its ability to enhance cap-dependent translation (43). Expression these findings suggest that TCF7L2 is involved in MALAT1- of the dominant negative 4EBP1-5A mutant profoundly repressed regulated glucose metabolism in cancer cells. expression of TCF7L2, as compared with vector control (Fig. 4D). To further confirm the importance of cap-dependent translation MALAT1 and TCF7L2 regulate gluconeogenesis through the in the TCF7L2 protein expression and MALAT1-mediated glyco- same pathway lytic effect, we ectopically expressed 4EBP1 wild type (WT) and To examine whether MALAT1 and TCF7L2 are regulating glu- mutant 4EBP1-4A (in which the four known phosphorylation coneogenesis through the same pathway, we knocked down both sites were replaced with alanine; ref. 44) in PHM-1 MALAT1 cells. MALAT1 and TCF7L2, either individually or together, in FLC4 Expression of the dominant negative 4EBP1-4A mutant pro- cells (Supplementary Fig. S7A and S7B). We examined gluconeo- foundly repressed expression of TCF7L2 compared with 4EBP1 genesis gene expression in these cells. We found that transient WT (Supplementary Fig. S4A) and repressed the expression of knockdown of both MALAT1 and TCF7L2, either individually or

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A 2.5 TCF7L2 2 ** TCF7L2 1.5 ** β-Catenin 1

Tubulin Relative intensity 0.5

PHM-1 0 Empty MALAT1 MALAT1 DMSO DMSO Rapamycin PHM-1 B 1.2 1.2 mTOR TCF7L2 1 1 mTOR 0.8 0.8 TCF7L2 0.6 *** 0.6 *** *** *** β-Actin 0.4 0.4 0.2 0.2 Tubulin Relative intensity PHM-1 0 0 Vector shmTOR#1 shmTOR#2 Vector shmTOR#1 shmTOR#2 PHM-1 PHM-1 C 1.2 1.2 Raptor TCF7L2 1 Raptor 1 TCF7L2 0.8 0.8 *** 0.6 ** 0.6 *** ** GAPDH 0.4 0.4 0.2 0.2 Relative intensity β-Tubulin 0 0 Vector shRaptor#1 shRaptor#2 Vector shRaptor#1 shRaptor#2 PHM-1 PHM-1 PHM-1

D 1.2 p4EBP1 1.2 TCF7L2 TCF7L2 1 1 0.8 0.8 p4EBP1 * *** 0.6 0.6 Total 4EBP1 0.4 0.4 0.2 0.2 Relative intensity GAPDH 0 0 Empty 4EBP1(5A) Empty 4EBP1(5A) β-Actin PHM-1 PHM-1

PHM-1

Figure 4. TCF7L2 translation is regulated by mTORC1-4EBP1. A, Left, Western blot analysis of PHM-1 cells transduced with lentivirus encoding either MALAT1 or an empty vector in the presence or absence (DMSO) of rapamycin. Right, quantification of TCF7L2 protein levels upon rapamycin treatment (n ¼ 4). B, Left, Western blot analysis of PHM-1 cells transduced with retroviruses encoding mTOR shRNAs. Right, quantification of mTOR and TCF7L2 protein levels upon shRNA knockdown (n ¼ 3). C, Left, Western blot analysis of PHM-1 cells transduced with retroviruses encoding Raptor shRNAs. Right, quantification of Raptor and TCF7L2 protein levels upon shRNA knockdown (n ¼ 4). D, Left, Western blot analysis of PHM-1 cells transduced with retroviruses encoding for empty vector pWZL-Hygro (empty) or PWZL-4EBP phosphorylation defective mutant (4EBP1-5A). Right, quantification of phosphorylated 4E-BP1 (n ¼ 2) and TCF7L2 protein levels upon overexpression of PWZL-4EBP1-5A (n ¼ 2). Error bars, SD. Student t test was used. , P < 0.05; , P < 0.01; , P < 0.001.

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A 1.2 SRSF1 Protein 1.2 TCF7L2 Protein 1 1

SRSF1 0.8 0.8 0.6 0.6 ** *** TCF7L2 Figure 5. 0.4 *** 0.4 *** SRSF1 regulates TCF7L2 translation. *** *** 0.2 0.2 A, Left, Western blot analysis of Relative intensity β-Actin HepG2 cells transduced with 0 0 lentiviruses containing shRNAs HepG2 against SRSF1 (shSF2#1, #2, #3) or an empty vector (shCon). b-Actin was used as loading control. Right, quantiﬁcation of SRSF1 and TCF7L2 B C protein levels upon shRNA 1.8 0.6 TCF7L2 mRNA ShControl ** knockdown (n ¼ 3). B, qRT-PCR of 1.6 0.5 ShSRSF1#1 * TCF7L2 mRNA levels in cells described in A. C. Relative 1.4 ShSRSF1#2 distribution of TCF7L2 mRNA 1.2 0.4 ** across the polysome fractions in 1 0.3 HepG2 cells transfected with either 0.8 ** control shRNA or shRNAs against SRSF1. Error bars, SD. Student t test 0.6 0.2 ** was used. , P < 0.05; , P < 0.01; 0.4 P < 0.1 , 0.001.

Relative expression 0.2

0 Fraction mRNA TCF7L2 0 Free Light Heavy Polysome fractionation

together, resulted in increased gluconeogenic gene expression down in FLC4 cells showed similar results (Supplementary Fig. without a significant additive effect (Supplementary Fig. S7C and S8C and S8D). These results suggest that TCF7L2 is required for S7D) without a significant additive effect. Similar results were the maintenance of the oncogenic properties of HCC cells. obtained in HepG2 cells (Supplementary Fig. S7E–S2H). These results suggest that MALAT1 and TCF7L2 modulate gluconeogen- Elevated nuclear expression of TCF7L2 in tumors from a mouse esis through the same pathway. model of HCC We next sought to determine whether in an in vivo mouse model TCF7L2 acts downstream of MALAT1 of HCC (Mdr2 / mice), which is known to upregulate To examine the potential of TCF7L2 as a downstream mod- MALAT1 (8), TCF7L2 is upregulated and if its expression corre- ulator of MALAT1, we transduced PHM-1 cells with lentiviruses lates with expression of genes controlling glycolysis and gluco- encoding either TCF7L2 or an empty vector (Fig. 6I and J). neogenesis. We examined TCF7L2 mRNA and protein expression Overexpression of TCF7L2 lead to enhanced lactate production in tumor and non-tumor inflamed liver samples from Mdr2 / (Fig. 6K). In contrast, transient knockdown of MALAT1 by siRNAs mice. Both Western blot analysis and immunohistochemistry in TCF7L2 overexpressing PHM-1 cells (Fig. 6L) did not show show that the protein expression of TCF7L2 was elevated in the significant changes in lactate production (Fig. 6M), suggesting tumor samples compared with the non-tumor liver samples, with that TCF7L2 acts as a downstream effector of MALAT1. nuclear localization in HCC tumors compared with adjacent parenchyma (Fig. 7F; Supplementary Fig. S9A). Similar to what TCF7L2 is required for MALAT1-mediated transformation was detected in cell lines, TCF7L2 mRNA levels were not signif- To examine whether TCF7L2 upregulation mediates MALAT1 icantly different in HCC tumors compared with non-tumor livers induced transformation, we knocked down TCF7L2 in PHM-1 (Supplementary Fig. S9B). Increased TCF7L2 protein levels was cells overexpressing human MALAT1 (Fig. 7A). Stable knockdown statistically significant (Supplementary Fig. S9C). Normalization of TCF7L2 in these cells resulted in decreased survival in a of protein to mRNA levels in these tumors suggests that there is clonogenic assay (Fig. 7B) and reduced formation of colonies in increased protein to mRNA ratios of TCF7L2 in most of the soft agar (Fig. 7C), demonstrating that cells overexpressing tumor samples compared with the normal samples (Supple- MALAT1 require TCF7L2 overexpression for their oncogenic mentary Fig. S9D). Furthermore, mRNA analysis by qRT-PCR properties. Knockdown of TCF7L2 in PHM-1 cells overexpressing showed upregulation of multiple glycolytic genes (Supplemen- human MALAT1 did not show a strong effect on the proliferative tary Fig. S9E), and downregulation of gluconeogenic genes capacity of these cells (Fig. 7D and E). To validate the importance (Supplementary Fig. S9F), in most of the tumor samples of TCF7L2 upregulation in HCC cells, we stably or transiently compared with normal samples. Taken together, our results knocked down TCF7L2 in HepG2 (Supplementary Fig. S8A) cells. suggest a potential role for MALAT1 in the regulation of TCF7L2 knockdown in these cells resulted in reduced formation glycolytic and gluconeogenic gene expression in HCC through of colonies in soft agar (Supplementary Fig. S8B). TCF7L2 knock- TCF7L2. This reveals yet another oncogenic function of

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LncRNA RNA MALAT1 Regulates Cancer Glucose Metabolism

A 1.2 TCF7L2 B 1.2 TCF7L2 1 1 qRT-PCR 0.8 * * 0.8 *** 0.6 TCF7L2 0.6 *** GAPDH 0.4 0.4 0.2 0.2 β-Actin Relative expression Relative intensity 0 0 PHM-1 MALAT1

Figure 6. C 1.2 D TCF7L2 mediates MALAT1 effects Lactate on glucose metabolism. A, Left, 1 Western blot of PHM-1 cells 0.8 *** overexpressing MALAT1 *** 0.6 transfected with siTCF7L2. Right, TCF7L2 quantiﬁcation of TCF7L2 protein 0.4 α-Tubulin levels upon TCF7L2 siRNA output Relative 0.2 treatment (n ¼ 2). B, qRT-PCR of β-Actin cells described in A. C, Extracellular 0 lactate production was measured in PHM-1 MALAT1 cells described in A using a lactate assay kit (n ¼ 3). D, Western blot analysis of PHM-1 cells overexpressing MALAT1 after EF1.2 1.2 GH1.2 1.2 GLUT1 transduction with lentiviruses 1 1 1 1 expressing TCF7L2 shRNAs. PKM2 0.8 0.8 0.8 ENO1 0.8 E–H, qRT-PCR of the indicated ** HK2 0.6 0.6 0.6 *** 0.6 ** genes in the glucose metabolic *** pathway in cells described in D.All 0.4 ** 0.4 ** 0.4 0.4 *** samples were normalized to 0.2 0.2 *** 0.2 0.2 GAPDH mRNA levels. I, Western

Relative expression 0 blot analysis of PHM-1 cells stably 0 0 0 expressing TCF7L2 or an empty vector. J, qRT-PCR of cells described in I. K, Extracellular lactate production was measured in 2.5 cells described in I using a lactate IJK80 Human TCF7L2 Lactate n ¼ ** assay kit ( 2). L, qRT-PCR of ** 2 PHM-1 cells overexpressing TCF7L2 60 and knocked down for MALAT1 by 1.5 siRNA. M, Extracellular lactate T7 40 1 production was measured in cells described in L using a lactate assay β-Actin 20 0.5 kit (n ¼ 2). Error bars, SD. Student output Relative Relative expression Relative t test was used. , P < 0.05; PHM-1 0 0 , P < 0.01; , P < 0.001. Empty TCF7L2 Empty TCF7L2

LM1.2 1.4 MALAT1 Lactate 1 1.2 1 0.8 0.8 0.6 *** 0.6 0.4 *** 0.4

0.2 Relative output 0.2 Relative expression 0 0 SiLuc SiMAL#1 SiMAL#2 SiLuc SiMAL#1 SiMAL#2 PHM-1 TCF7L2 PHM-1 TCF7L2

MALAT1, promoting the "Warburg effect" and repressing glu- lying molecular mechanisms leading to this phenomenon remain coneogenesis during the development of human HCC. unclear in many tumors. The present study reveals an unexpected function of lncRNA MALAT1 in promoting aerobic glycolysis and repressing gluconeogenesis in HCC, adding to its previously Discussion known oncogenic activities (8, 49, 50). Our initial observation Increased aerobic glycolysis, or the "Warburg effect," is one of that MALAT1 overexpression in PHM-1 cells changes the color of the ﬁrst identiﬁed hallmarks of cancer (32). However, the under- the cell medium, led us to explore the role of this oncogenic

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A B 1.2 Empty 1

0.8 MALAT1 TCF7L2 0.6 * ** β-Tubulin 0.4 MALAT1 shTCF#1 0.2 β-Actin Relative intensity MALAT1 PHM-1 MALAT1 0 shCon shTCF#1 shTCF#2 shTCF#2 PHM-1 MALAT1 PHM-1 D E C 2.5 30 80 shTCF#1 *** Empty 70 shTCF#2 25 MALAT1 shCon 2 60 20 50 1.5 15 40 * colonies 1 30 10 20 0.5 Relative no. of Agar Agar of Relative no. 5 Relative absorbance Relative absorbance ** Relative absorbance 10 0 0 0 Empty MALAT1 MALAT1 MALAT1 024487296 024487296 shTCF#1 shTCF#2 Time (h) Time (h) PHM-1

F TCF7L2 TCF7L2

P T

T P

G mTORC1 SRSF1 MALAT1

p-4EBP WNT Signaling TCF7L2 Gluconeogenesis

Gluconeogenesis Glycolysis

Hepatocellular carcinoma

Figure 7. TCF7L2 is required for MALAT1-induced transformation. A, Left, Western blot of PHM-1 cells overexpressing MALAT1 transduced with lentiviruses encoding TCF7L2 shRNAs. Right, quantiﬁcation of TCF7L2 protein levels upon TCF7L2 knockdown by shRNA (n ¼ 2). B, Clonogenic assay of control PHM-1 cells stably expressing empty vector and cells described in A. C, Growth in soft agar assay of control PHM-1 cells stably expressing empty vector and cells described in A. Colonies were counted 28 days after seeding. Error bars, SD (n ¼ 3). Two tailed Student t test was used. , P < 0.05; , P < 0.01; , P < 0.001. D, Proliferation assay of PHM-1 cells stably expressing hMALAT1 or an empty vector. E, Proliferation assay of cells described in A. F, Immunohistochemistry staining for TCF7L2 in liver tumor (T) specimens, including surrounding parenchymal (P) tissue from two 12-months-old Mdr2/ mice (scale bar, 50 mm). Note the enhanced nuclear TCF7L2 staining in malignant hepatocytes. G, Scheme summarizing the role of MALAT1 in regulating glucose metabolism in HCC. Red lines represent pathways by which MALAT1 regulates glucose metabolism in HCC as described here. Blue lines represent pathways described by others.

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lncRNA in facilitating aerobic glycolysis. Indeed, we found a TCF7L2 as measured by luciferase activity without an effect on functional correlation between MALAT1 expression, glucose luciferase mRNA levels (Fig. 3). secretion and lactate production in PHM-1 cells overexpressing Signaling by the PI3K/AKT/mTOR pathway profoundly affects MALAT1 and HCC cells (Figs. 1 and 2). Using flow cytometry, we mRNA translation through phosphorylation of downstream tar- demonstrate glucose uptake at a single cell level, eliminating the gets, such as 4EBP1 and S6K1 (54). The cap-dependent protein possibility that it is due to cell density or cell proliferation. synthesis pathway serves as a pleotropic integrator and amplifier Furthermore, MALAT1 overexpression in HCC cells increased of many essential oncogenic signals (53, 55). Our data show that the expression of glycolytic genes and its knockdown resulted TCF7L2 is specifically regulated by the mTORC1–4EBP1 axis in increased expression of gluconeogenic genes (Figs. 1 and 2; (Fig. 4). Because TCF7L2 is a major transcriptional regulator of Supplementary Fig. S2). the Wnt pathway, it is possible that the mTORC1 pathway, Gluconeogenesis is a process that consumes energy to regen- through its regulation of TCF7L2 translation, can modulate the erate glucose in the liver, secreting it to the blood when blood Wnt pathway. The crosstalk between these two signaling pathways glucose levels drop (34). In this pathway three specific steps, has not been demonstrated to our knowledge. Next, to further catalyzed by gluconeogenesis enzymes, are used to bypass the substantiate the regulation of cytoplasmic TCF7L2 translation by irreversible reactions of glycolysis. In this regard, simultaneous nuclear MALAT1, we looked at the regulation of TCF7L2 by SRSF1. activation of both pathways may result in a futile cycling of SRSF1 is a nuclear splicing factor and MALAT1 was shown to glucose that is detrimental to cell survival (51). To avoid such regulate the expression and function of SRSF1. SRSF1 was shown futility, activation of either pathway should be mutually exclusive. to activate mTOR and protein translation (45, 46, 56). Indeed, we Results of our experiments, with knockdown of MALAT1 in HCC found that SRSF1 knockdown reduced TCF7L2 translation in cell lines, demonstrate increased expression of gluconeogenic HepG2 cells (Fig. 5). This result suggests that increased protein genes. Considering the essential requirement of energy and build- expression of TCF7L2 by MALAT1 could be, in part, attributed to ing blocks for cell doubling, it is evident that upregulation of increased expression of SRSF1 by MALAT1. This result also pro- gluconeogenesis, an energy consuming process, would result in vides an explanation for the reduced expression of TCF7L2 in the the suppression of HCC proliferation. This is indeed the case as presence of Rapamycin, as knockdown of SRSF1, which is known knockdown of MALAT1 in HCC cell lines resulted in decreased to activate mTORC1, in PHM-1 and HepG2 cells resulted in proliferation (8). As gluconeogenesis is dramatically impaired in reduced protein expression of TCF7L2 (Figs. 4 and 5). malignant hepatocytes, similar to what we observed in Mdr2 / The result of increased expression of gluconeogenesis genes liver tumor samples compared with adjacent normal liver paren- upon TCF7L2 knockdown is in agreement with previous studies chyma, it is possible that gluconeogenesis represents a metabolic where it was shown that TCF7L2 is a negative regulator of barrier to HCC development. This is the first report showing the gluconeogenesis (36, 47). Regarding glycolytic gene expression regulation of gluconeogenesis by MALAT1 in HCC. Importantly, regulation by TCF7L2, this is the first description of the regulation we also establish regulation of various glycolytic genes (GLUT1, of glycolytic gene expression by TCF7L2 in HCC. HK2, ENO1, and PKM2) by MALAT1 in PHM-1 cells. To maintain As expected from the alteration in glycolytic and gluconeogenic the survival and rapid proliferation, cancer cells normally elevate enzyme expression, overexpression of TCF7L2 resulted in expression of glycolytic genes. Several oncoproteins and tumor increased lactate production while knockdown of TCF7L2 suppressors were found to regulate enzymes that facilitate glyco- resulted in decreased lactate production (Fig. 6). These results lytic tumor glucose metabolism. In this study, we report for the suggest direct regulation of cancer glucose metabolism by first time that lncRNA MALAT1 regulates an array of glycolytic TCF7L2. Furthermore, knockdown of MALAT1 in TCF7L2-over- genes in HCC. expressing cells did not change lactate production, suggesting that Gluconeogenesis has been shown to be negatively regulated TCF7L2 acts downstream to MALAT1. by TCF7L2 in various studies (36, 47). Even though there are no Because TCF7L2 regulates glucose metabolism and acts studies showing the role of TCF7L2 in glycolysis or the "War- downstream of MALAT1, we sought to examine the importance burg effect," TCF7L2 has been implicated in HCC in various of TCF7L2 in MALAT1-mediated oncogenic activity. To this end studies (48) and has been shown to be an important mediator we knocked-down TCF7L2 in PHM-1 cells overexpressing of the Wnt signaling pathway. Wnt signaling has been shown to MALAT1. Stable knockdown of TCF7L2 in these cells resulted modulate the "Warburg effect" (15). This prompted us to look in decreased survival in a clonogenic assay and reduced for- at the regulation of TCF7L2 by MALAT1. The elevation in mation of colonies in soft agar (Fig. 7). Furthermore, knock- TCF7L2 protein levels upon MALAT1 overexpression, as well down of TCF7L2 in HCC cell lines (HepG2 and FLC4) resulted as the decrease in TCF7L2 protein levels upon MALAT1 knock- in reduced oncogenic properties as seen by reduced formation down, results from translational regulation and is not a result of colonies in soft agar (Supplementary Fig. S8). These results of changes in transcription or stability (Fig. 3). Control of suggested that TCF7L2 expression is essential for MALAT1- mRNA translation constitutes a critical step in the regulation mediated transformation. TCF7L2 overexpression in HCC has of gene expression and in cancer (52, 53). Polysome profiling been reported in several studies (48). Our western blot and of TCF7L2 mRNA showed increased translation of TCF7L2 in gene expression analysis on Mdr2 / mice liver tumor samples MALAT1-overexpressing cells while reduced translation of revealed strong upregulation of MALAT1 at the RNA level and TCF7L2 in MALAT1 knockdown cells (Fig. 3). Translation TCF7L2 at the protein level (Fig. 7; Supplementary Fig. S9). initiation efficiency can be regulated by the 50UTR. Secondary Furthermore, Mdr2 / liver tumor samples showed elevated RNA structures at the 50UTR of many mRNAs inhibit translation glycolytic and repressed gluconeogenic gene expression (Sup- initiation and this inhibition can be alleviated by RNA heli- plementary Fig. S9), suggesting a significant relevance of cases, which are recruited by the eIF4G–eIF4E complex (40). MALAT1-mediated tumor glucose metabolism in the develop- Knockdown of MALAT1 resulted in reduced 50UTR activity of ment of tumors in vivo.

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It is important to note that the true clinical implications of these Authors' Contributions results needs to be examined further in human normal liver and Conception and design: P. Malakar, R. Karni HCC clinical samples. Development of methodology: P. Malakar Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P. Malakar, I. Stein, A. Saragovi, R. Winkler, Conclusion N. Stern-Ginossar, M. Berger, E. Pikarsky Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Taken together, our data suggest that MALAT1 acts as a regulator computational analysis): P. Malakar, I. Stein, A. Saragovi, R. Winkler, M. Berger, of glucose metabolism in HCC. Our results add insight to the R. Karni mechanisms of cancer glucose metabolism and cancer progres- Writing, review, and/or revision of the manuscript: P. Malakar, R. Karni sion. The novel ﬁndings from the present study, together with the Study supervision: R. Karni signiﬁcant discoveries from previous studies, place MALAT1 at the crossroad of cellular metabolism and carcinogenesis (Fig. 7G). Acknowledgments MALAT1 regulates the expression of TCF7L2 at the translational The authors wish to thank Dr. Zahava Siegfried for comments on the article level. TCF7L2 regulation by MALAT1 is through a mTORC1- and Fatima Gebauer (CRG, Barcelona) for the pSG5 Luc Plasmid. This study dependent pathway via cap-dependent translation. TCF7L2 plays was supported in part by Israel Science Foundation (ISF; ISF Grant no. 1290/12 an important role in MALAT1-induced tumorigenesis and altered to R. Karni). glucose metabolism in HCC development. These results point toward the fact that knockdown of MALAT1 or reduction of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked TCF7L2 levels might serve as new strategies based on tumor advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate glucose metabolism for the treatment of HCC. this fact.

Disclosure of Potential Conflicts of Interest Received May 10, 2018; revised January 10, 2019; accepted March 20, 2019; No potential conflicts of interest were disclosed. published first March 26, 2019.

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LncRNA RNA MALAT1 Regulates Cancer Glucose Metabolism

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www.aacrjournals.org Cancer Res; 79(10) May 15, 2019 2493

Long Noncoding RNA MALAT1 Regulates Cancer Glucose Metabolism by Enhancing mTOR-Mediated Translation of TCF7L2

Pushkar Malakar, Ilan Stein, Amijai Saragovi, et al.

Cancer Res 2019;79:2480-2493. Published OnlineFirst March 26, 2019.

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